Diverse environmental stresses including hypoxia, oxidative stress, viral infection, nutrient limitation, radiation, ischemia, and others. Such stresses may affect a variety of tissues, including membranes, cytoplasm, the endoplasmic reticulum (ER), and the like. Stress to the ER has been reported to harm the efficient functioning of protein folding and cellular activities (Harding, H. P., et al., “Transcriptional and translational control in the mammalian unfolded protein response,” Annu. Rev. Dev. Biol. 18:575-599 (2002); Szegezdi, E., et al., “Mediators of endoplasmic reticulum stress-induced apoptosis,” EMBO Rep. 7: 880-885 (2006); Wek, R., et al., “Translational control and the unfolded protein response,” Antioxidants and Redox Signaling 9:1-15 (2007)). In order to alleviate cellular injury or, conversely, initiate apoptotic cell death, cells induce the ISR (Harding, H. P., et al., “An integrated stress response regulates amino acid metabolism and resistance to oxidative stress,” Mol. Cell. 11: 619-633 (2003)). The ISR evokes translational reprogramming as its primary consequence and secondarily affects the transcriptional profile of cells. ER stress is one type of ISR induced stress (Ron, D., “Translational control in the endoplasmic reticulum stress response,” J. Clin. Inv. 110:1383-1388 (2002); Boyce, M., et al., “Cellular response to endoplasmic reticulum stress: a matter of life or death,” Cell Death Differ. 13:363-373 (2006)). The foregoing publications and all other additional publications cited herein are incorporated herein by reference.
Stress to the ER has been implicated in type 2 (non-insulin dependent) diabetes, Wolfram Syndrome, tyrosinemia, cystic fibrosis, tumor growth under hypoxic conditions, cerebral ischemia, neurodegenerative diseases (Familial Amyotrophic Lateral Aclerosis, Familial Alzheimer's disease, Huntington's disease), and others. However, whether this stress to the ER is a primary cause of diseases or only a secondary pathological phenomenon is currently being debated (Zhao, L., et al., “Endoplasmic reticulum stress in health and disease,” Current Opinion Cell Biol. 18:444-452 (2006)). In skeletal diseases that are linked to stress to the ER, four specific genetic disorders are listed in “Online Mendelian Inheritance in Man (OMIM)” database.
One such genetic disorder, Wolcott-Rallison Syndrome, is an autosomal recessive disorder characterized by epiphyseal dysplasia, osteoporosis and permanent neonatal or early infancy insulin-dependent diabetes. In two unrelated patients with Wolcott-Rallison syndrome, two mutations in the EIF2AK3 gene (PERK) were identified. Another disorder, Synovial cell hyperplasia, results in destruction caused by mutations in SYVN1 (synovial apoptosis inhibitor 1; HRD1). SYVN1 is a ubiquitin ligase whose expression is induced by the unfolded protein response following stress to the ER (Kaneko M, et al., “Human HRD1 protects against ER stress-induced apoptosis through ER-associated degradation,” FEBS Lett. 532:147-52 (2003)). Expression of HRD1 protects cells from apoptosis by inducing degradation of abnormally processed proteins that accumulate in the ER. Another disorder, Inclusion Body Myopathy with Paget Disease of Bone and Frontotemporal Dementia (IBMPFD), is characterized by adult-onset proximal and distal muscle weakness (clinically resembling a limb-girdle muscular dystrophy syndrome), early-onset Paget Disease of Bone, and premature frontotemporal dementia (FTD). IBMPDF is caused by mutation in VCP (valosin-containing protein), which is required for the export of ER proteins into the cytosol (Ye, Y., et al., “The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol,” Nature 414:652-656 (2001)). Cardiac failure and cardiomyopathy have been observed in later stages. Paget Disease of Bone involves focal areas of increased bone turnover that typically lead to spine and/or hip pain and localized enlargement and deformity of the long bones. Another disorder, Marfan Syndrome, is characterized by disproportionately long limbs and digits, anterior chest deformity, mild to moderate joint laxity, and vertebral column deformity (scoliosis and thoracic lordosis) (Sponseller, P. D., et al., “The thoracolumbar spine in Marfan syndrome,” J. Bone Joint Surg. 77-A:867-876 (1995)). This syndrome is caused by missense mutations in FBN1 (fibrillin-1), which increases retention of mutated products in the ER (Whiteman, P., et al., “Defective secretion of recombinant fragments of fibrillin-1: implications of protein misfolding for the pathogenesis of Marfan syndrome and related disorders,” Hum. Molec. Genet. 7:727-737 (2003)).
Without being bound by theory, it is believed herein that genetic diseases, such as Wolcott-Rallison syndrome, synovial cell hyperplasia, IBMPFD (Inclusion Body Myopathy associated with Paget disease of bone and Frontotemporal Dementia), and Marfan syndrome are directly or indirectly linked to the biological process of coping with stress to the ER, a form of integrated stress response (ISR) where translational regulation plays a key role. IBMPFD and Marfan syndrome are linked to stress to the ER, and in particular are linked to abnormal retention of mutated proteins in the ER. In Wolcott-Rallison syndrome the responses to the ER stress are impaired by the kinase for phosphorylation of eIF2α, while in mutation in SYVN1 bone destruction is triggered by insufficient protection of cells from ER stress-induced apoptosis. In addition, but without being bound by theory, it is believed herein that many skeletal diseases, including bone disorders, diseases, and even complications arising from bone injury are directly or indirectly linked to the biological process of coping with stress to the ER. One such bone disease is osteoporosis, which is most common in women after menopause but may also develop in elderly men. Osteoporosis is a bone disease that reduces bone mass and strength. Because of its risk of fracture in the femoral neck and long bones, it significantly affects quality of life.
Although the role of stress to the ER may differs in those diseases, though without being bound by theory, it is believed herein that active intervention in regulating stress to the ER and other ISR with pharmacological agents, with or without mechanical stimulation, will elevate anabolic responses in vitro and in vivo.
Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) is a highly conserved regulatory event activated during ISR (Ron, D., et al., “eIF2α phosphorylation in cellular stress responses and disease,” Translational Cont. Biol. Med. 13:349-372 (2007)). ISR-driven phosphorylation on serine 51 of eIF2α blocks an exchange process of eukaryotic translation initiation factor 2B (eIF2B) from GDP-bound eIF2 to GTP-bound eIF2 (Proud, C. G., “eIF2 and the control of cell physiology,” Semin. Cell Dev. Biol. 16: 3-12 (2005)). Consequently, the global translation-initiation is suppressed except for a group of specific genes whose expression is crucial for an adaptive response for survival.
One such gene translationally activated in ISR is the transcriptional regulator activating transcription factor, ATF4. ATF4 mRNA consists of two upstream open reading frames (uORF) (uORF1 and uORF2) together with the ATF4-coding region (Vattem, K. M., et al., “Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells,” Proc. Natl. Acad. Sci. U.S.A. 101:11269-11274 (2004)). When eIF2-GTP is abundant in non-stressed cells, ribosomes scanning downstream of uORF1 reinitiate translation at uORF2 that aborts translation of ATF4 protein. During the ISR process, reduction in the levels of eIF2-GTP promotes non-commitment of ribosomes at uORF2 and increases re-initiation of translation at the ATF4-coding region.
ATF4 is also an essential regulator in bone development and its deficiency in transgenic mice results in a phenotype having delayed bone formation as well as low bone mass (Yang, X., et al., “ATF4 is a substrate of RSK2 and an essential regulator of osteoblasts biology: implication for Coffin-Lowry syndrome,” Cell 117:387-398 (2004)). In addition to ATF4, it is believed herein that other ISR-linked genes may be important in bone development, including ATF3 and CHOP. Further, genes that are involved in bone remodeling, including Runx2, Osterix, and Rank 1, may be also affected by active intervention in regulating stress to the ER and other ISR.
A number of pharmacological agents are known to promote phosphorylation of eIF2α, including thapsigargin (Wek, R., et al., “Translational control and the unfolded protein response,” Antioxidants and Redox Signaling 9:1-15 (2007)) and tunicamycin. MC3T3 mouse osteoblast-like cells have been incubated with thapsigargin (C34H50O12; M.W. 651) and tunicamycin (C39H64N4O16; M.W. 840) (Hamamura, K., et al., “Stress to endoplasmic reticulum of mouse osteoblasts induces apoptosis and transcriptional activation for bone remodeling,” FEBS Lett. 381:1769-1774 (2007)). Although both agents activate PERK, their mechanisms are different. Thapsigargin raises a cytosolic calcium concentration by blocking Ca++ pumps, while tunicamycin is an inhibitor of N-linked glycosylation and the formation of N-glycosidic protein-carbohydrate linkages.
It is suggested herein that there are two ways for pharmacological agents to regulate phosphorylation of eIF2α, as inhibitors of dephosphorylation and as inducers of phosphorylation. Described herein are compounds, compositions, and methods useful for inhibition of dephosphorylation. Salubrinals are one such family of compounds capable of acting as inhibitors of dephosphorylation. The parent compound salubrinal (C21H20Cl3N4OS; M.W. 479.8) has been reported to protect the rat pheochromocytoma cell line from ER stress-induced apoptosis (Boyce, M., et al., “A selective inhibitor of eIF2α dephosphorylation protects cells from ER stress,” Science 307: 935-939 (2005)). In addition, the effect of salubrinal was examined using a rat neuronal injury model in which acute ER stress was induced by the glutamate receptor agonist kainic acid. The results revealed that intra-cerebroventricular or intra-peritoneal administration decreased excitotoxic neuronal death in vivo (Sokka, A. L., et al., “Endoplasmic reticulum stress inhibition protects against excitotoxic neuronal injury in the rat brain,” J. Neurosci. 27: 901-908 (2007)). Furthermore, administration of salubrinal to brainstem motoneurons in mice suppressed motoneuronal injury in response to hypoxia and reoxygenation events (Zhu, Y., et al., “eIF-2α protects brainstem motoneurons in a murine model of sleep apnea,” J. Neurosci. 28: 2168-2178 (2008)). Direct injection into a mouse hippocampus modulated ATF4-dependent long-term synaptic plasticity and memory (Costa-Mattioli, M., et al., “Translational control of hippocampal synaptic plasticity and memory by the eIF2alpha kinase GCN2,” Nature 436:1166-1173 (2005); Costa-Mattioli, M., et al., “eIF2alpha phosphorylation bidirectionally regulates the switch from short- to long-term synaptic plasticity and memory,” Cell 129:195-206 (2007)).
In contrast, the potential benefits of salubrinal to β-cells is reportedly controversial. While β-cell loss in type 1 diabetes is an autoimmune-mediated process and a linkage to ER stress is yet to be examined, type 2 diabetes results from a reduced ability of β-cells to secrete enough insulin to stimulate glucose utilization. Thus, accumulating evidence indicates that stress to the ER plays a role in β-cell dysfunction and death in type 2 diabetes (Eizirik, D. L., et al., “The role for endoplasmic reticulum stress in diabetes mellitus,” Endocrine Reviews 29:42-61 (2008)). However, administration of 5-75 μM salubrinal induced apoptosis in rat β-cells in a dosage dependent manner, demonstrating that excessive eIF2α phosphorylation is poorly tolerated by β-cells (Cnop, M., et al., “Selective inhibition of eukaryotic translation initiation factor 2 alpha dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic beta-cell dysfunction and apoptosis,” J. Biol. Chem. 282: 3989-3997 (2007)). Thus, without being bound by theory, it is believed herein that the effects of salubrinal are dependent both on dosage and cell type. Further, it is believed herein that in β-cells it is possible that phosphorylation of eIF2α plays a dual role, acting as beneficial regulator of insulin production or as trigger of dysfunction and apoptosis. Alternatively, salubrinal may have toxic side effects unlinked to eIF2α phosphorylation in β-cells and other specific cells. Also, but without being bound by theory, it is believed herein that strong and/or prolonged exposure to stress also leads to apoptosis.
Though the consequences of promoting and/or maintaining phosphorylation of eIF2α are still being investigated, it has been discovered herein that inhibition of one or more phosphatase complexes by salubrinal, and derivatives thereof, thus promoting and/or maintaining phosphorylation of eIF2α, may represent a viable method for treating integrated stress response-induced apoptosis, including ER stress-induced apoptosis. Accordingly, bone diseases, such as osteoporosis, bone defects, bone injuries, such as fractures, and other bone conditions, arising from or exacerbated by such induced apoptosis may be treated using the compounds, compositions, and methods described herein.
In one illustrative embodiment of the invention, compounds, pharmaceutical compositions thereof, and methods for using each are described herein for inhibiting phosphatase complexes in treating diseases that arise from apoptosis of certain cell populations. In one aspect, the compounds, pharmaceutical, and methods are described for treating integrated stress response-induced apoptosis. It is appreciated herein that a wide variety of diseases in a wide variety of tissues may be caused by integrated stress response-induced apoptosis. In another embodiment, compounds, compositions, and methods are described herein for treating diseases that arise from endoplasmic reticulum stress-induced apoptosis. In another embodiment, compounds, compositions, and methods are described herein for inhibiting the dephosphorylation of, maintaining the phosphorylation of, and/or promoting the phosphorylation of eIF2α.
In another illustrative embodiment, compounds, compositions, and methods are described herein for inhibiting one or more phosphatase complexes, wherein the inhibition prevents eIF2α dephosphorylation at the endoplasmic reticulum of the cell below a threshold where apoptosis might otherwise occur. In one variation, the inhibition results in or also results in maintaining the eIF2α phosphorylation at the endoplasmic reticulum of the cell above a threshold level where apoptosis might otherwise occur. In another variation, the inhibition results in or also results in stabilizing the eIF2α phosphorylation state of the endoplasmic reticulum of the cell above a threshold level where apoptosis might otherwise occur.
In another embodiment, compounds, compositions, and methods are described herein for treating a bone disease, bone, bone injury, and/or bone defect. Illustrative diseases, disorder, injuries, defects, and the like include, but are not limited to, osteoporosis, osteopenia, fracture, surgical wounds, spinal bone defects, osteonecrosis, pediatric hip necrosis, osteonecrosis of the jaw bone, bone defects or degradation arising from cancer treatment, including chemotherapy, radiation therapy, and the like.
In another embodiment, the compounds, compositions, and methods described herein include one or more salubrinals, including salubrinal, and analogs and derivatives of salubrinal, and/or pharmaceutically acceptable salts thereof, hydrates thereof, and/or solvates thereof, and/or a prodrug of any of the foregoing. It is to be understood, that as used herein, the term “salubrinal” may refer both to the individual compound as well as the family of compounds that include such analogs and derivatives. In another embodiment, the salubrinals are of the formula
or a pharmaceutically acceptable salt thereof, wherein:
X and Y are independently O or S;
R1 is alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, or heteroaryl, each of which is optionally substituted;
R2 is alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, cycloalkylalkenyl, cycloalkenylalkenyl, arylalkenyl, or heteroarylalkenyl, each of which is optionally substituted;
Ra is optionally substituted alkyl;
Rb is H or optionally substituted C1-C6 alkyl;
Rc, Rd, and Re are each independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, acyl, or a prodrug capable of releasing the attached nitrogen in vivo to form the corresponding H or salt derivative thereof;
and A and B are independently H, or optionally substituted C1-C6 alkyl.
In another embodiment, a therapeutically effective amount of a compound described herein, such as salubrinal, or an analog or derivative thereof, is administered to a patient in need of relief from a bone disease, injury, or defect. In another embodiment, a therapeutically effective amount of a compound described herein, such as salubrinal, or an analog or derivative thereof, is included in a pharmaceutical composition for treating a patient in need from a bone disease, injury, or defect. In another embodiment, a therapeutically effective amount of a compound described herein, such as salubrinal, or an analog or derivative thereof, is used in the manufacture of a medicament for treating a patient in need from a bone disease, injury, or defect.